The photocleavable compounds, the so called “caged reagents” are very useful compounds in the course of investigation of biological processes. Such reagents and the use thereof are described particularly in the description of EP 1 757585 A1 and U.S. Pat. No. 6,765,014 by the inventors and in the articles of Morrison et al. (Photochemical & Photobiological Sciences, (2002), 1, 960), M. Canepari et al. (J. Neurosci Methods, (2001), 112, 29), M. Canepari et al. (J. Physiol., (2001), 533, 765). These compounds are biologically inactive but due to light these compounds cleave and biologically active compounds evolve. These compound evolve fast due to UV or visible light. These photocleavable compounds can be used for delivering the biologically active compounds, such us neuroactive amino acids to the place where their activity is necessary. The release of active compounds from photocleavable compounds using one- or multi-photon irradiation method, preferably two-photon irradiation method is a dynamically developing method of researches of neurons as it is described by Fedoryak et al. in their article (Chemical Communications, (2005), 29, 3664-3666). In the course of a multi-photon irradiation process the photochemical reaction is carried out by excitation of two or more photons. With the use of these methods the stimulation of neurons can be carried out locally and very specifically. The above-mentioned documents disclose the release of glutamic acid and gamma-amino-butiric acid (GABA) from photocleavable derivatives thereof using mono- or two-photon irradiation processes. The photocleavable compounds were used as bases in all cases. In the published patent application No. EP 1757585 and in the U.S. Pat. No. 6,765,014 7-indoline compounds are described which are used as photocleavable reagents. In these documents the photocleavable compounds are described as bases or salts formed with cations. In the publication of the patent application No. WO2008/094922 di-nitro derivatives are described as base or salts with cations.
The [2-amino-5-(4-methoxy-7-nitro-2,3-dihydro-indol-1-yl)-5-oxo-pentanoic acid (hereinafter as “MNI-Glu”) is a mononitroindoline (MNI) derivative which contains a glutamic acid component and is a well known and useful compound for the multi-photon irradiation examinations of the nervous system. However, the quantum yield of these mono-nitro-indoline derivatives is low and the release of active amino acids is also relatively slow and the maximum of their photon absorption is also not ideal. A higher quantum yield can be achieved with the use of dinitro-indol derivatives, such as the compounds based on the structure of the 4-methoxy-5,7-dinitro-2,3-dihydro-1H-indol. Such compounds are e.g. the 2-amino-5-(4′-methoxy-5′,7′-dinitro-2′,3′-dihydro-indol-1-yl)-5-oxo-pentanoic acid (hereinafter as “DNI-Glu”) which is disclosed by Fedoryak et al. (Chemical Communications, (2005), 29, 3664-3666), G. C. R. Ellis-Davies et al. (The Journal of Neuroscience, Jun. 20, (2007), 27(25), 6601-6604) and G. Papageorgiou et al. (Photochemical & Photobiological Sciences, (2005), 4(11), 887-896). The biological experiments are not disclosed in these articles but only the preparation of the analogue compounds is disclosed. The preparation of DNI-Glu according to Fedoryak et al. is carried out by a two-step nitration. At first the 2-amino-5-(4-methoxy-2,3-dihydro-indol-1-yl)-5-oxo-pentanoic acid which was protected on its amino and carboxyl groups was prepared then nitrated at the position 7. The mono-nitro derivative was nitrated at the position 5 in the next step. Subsequently, the protecting groups were removed, thus the product named DNI-Glu was obtained. A similar process is described in the patent application No. WO2008/094922. According to the inventors the effective process for the preparation of DNI-Glu requires an appropriate purity of the unprotected mono-nitro amino acid for the second nitration step. Thus the second nitro group can be introduced only in a second step. It was found that the dissolution of DNI-Glu is so bad in physiological solutions that in two-photon irradiation experiments of hippocampus neurons no electrical signals had been arisen. A further problem was that the DNI-Glu separated from the frozen buffer and could not be dissolved when it was warmed up to room temperature. These problems were eliminated by the insertion of a carboxy-methoxy group at the position 4 of the DNI-Glu according to the patent application. The 4-carboxy-methoxy-5,7-dinitrindolinyl-Glu (hereinafter CDNI-Glu) was prepared by a two-step nitration process. The CDNI-Glu compound was prepared only with a yield of 6.9% based on the indol derivative. Thus, according to the prior art the much better quantum yields of DNI derivatives could not be utilized because such stable derivatives could not be prepared which were appropriate stable either during storage or during the biological tests. Thus these reagents could not be prepared on industrial scale because their industrial manufacture and the preservation of their stability were not solved. Furthermore, there was no solution for the elimination of the interferences in the tests caused by biologically active compounds which spontaneously formed from easily degradable DNI derivatives. The known photochemically cleavable compounds, the so-called “caged” compounds applicable in examination processes were used only in the form of a base, but some of them were prepared also as a metal salt. These compounds have several disadvantageous properties. In the form of a base it is difficult to handle them, they are usually hygroscopic, light sensitive and labile. The photocleavable compounds hydrolyse spontaneously in the conditions of the experiments in many cases and the releasing active compounds interfere the experiment. It is a known fact that the neuroactive amino acids and amines are released quickly during the biological processes. Therefore the so-called “caged” compounds can be a good model of the physiological processes which degrade rapidly by the effect of irradiation. But these compounds more and more tend to hydrolyse during the experiment as well, therefore a considerable amount of amino acid or neuroactive amine gets into the measuring media, in the intercellular fluidum among the neurons before the irradiation. This is especially true for the DNI compounds which have a high quantum yield. Above a certain threshold these compounds can sensibilize the neurons, give a false result of the experiment, and in a higher concentration they may even cause the death of the examined neurons.
It has been a need for the preparation of such photocleavable compounds or the change of the used experimental conditions enabling the elimination of the above-mentioned disadvantages, so the cleavage of the “caged” compounds be fast, but the compounds resulted of the spontaneous hydrolysis do not disturb the measurement.